Cell culture systems of animal cells, in particular mammalian cells (including human cells), are important for the production of many important (genetically engineered) biological materials such as vaccines, enzymes, hormones and antibodies. The majority of animal cells are anchorage-dependent and require attachment to a surface or cell culture support for their survival and growth.
Cell attachment also plays an important role in medical applications such as wound treatment (including artificial skin materials, but also sticking plasters or band-aids comprising compounds or compositions that promote healing and cell attachments), bone and cartilage (re)growth and implantations and artificial blood vessel materials. Thus in medical applications often the demand is that a material, such as an implant or transplant material, comprises a biocompatible coating in terms of cell attachment.
Another area of interest in relation to cell attachment is the blocking of attachment receptors of cells. For instance by blocking the attachment receptors cancer metastasis may be influenced or inhibited, platelet aggregation may be influenced in antithrombotic compositions and tissues adhesion may be prevented, e.g. after surgery, or may be promoted, e.g. for dental products or other medical products.
In US 2006/0241032 RGD-enriched gelatin-like proteins with a minimum (increased) level of RGD motifs and with a certain distribution of said RGD motifs are disclosed that were found to be highly suitable for cell adhesion and cell binding in medical and biotechnological applications. The cell binding peptides described therein have good cell attachment properties.
However, susceptibility to degradation has been a limiting factor in the ability to produce large amounts of recombinant gelatins.
EP 926543 and Werten et al. 1999 (Yeast 15, 1087-1096) describe a production method of recombinant gelatins, wherein high yields of non-hydroxylated fragments of the helical domain (consisting of Gly-Xaa-Yaa triplet repeats) of mouse type I (encoding a 21 kDa and 28 kDa, calculated mw, COL1A1 peptide and a 53 kDa COL1A2) and rat type III (COL3A1) are produced in the methylotrophic yeast Pichia pastoris. Yields were between 3-4 g/liter clarified broth and up to 14.8 g/liter for a multicopy transformant of COL3A1. For COL3A1 degradation could be reduced by changing the fermentation pH from pH 5 to pH 3. In contrast, this did not reduce degradation of COL1A1, which appeared to contain an endopeptidase motif (Met-Gly-Pro-Arg). Upon site-directed mutagenesis of this motif to Arg-Gly-Pro-Met, degradation was reduced and it was speculated that a Kex2 protease or a Kex2-like protease recognized the motif [Leu-Ile-Val-Met]-Xaa-Yaa-Arg. However, yields of COL1A1 sequences still need to be improved. Werten et al. hypothesize that the yield of COL1A1 remains below that of COL3A1 due to the codon usage of COL1A1 being different from that of highly expressed P. pastoris genes, while the codon usage of COL3A1 is similar to that of highly expressed P. pastoris genes.